The initiation of DNA replication is a critical control point of the cell cycle, since abnormal replication initiation leads to genome instability, cancer and other inherited diseases. Accurate control of initiation requires the ordered assembly of prereplication protein and preinitiation protein complexes at origins of replication. We have identified a novel DNA unwinding element binding protein, DUE-B, which binds specifically to the essential DUE of the human c-myc replication origin and other origins. DUE-B is necessary for normal entry into the DNA synthetic S phase of the cell cycle and for the initiation of replication. DUE-B functions after formation of the prereplication complex but before origin template unwinding, and is required to load the preinitiation complex proteins Cdc45 and TopBP1 at origins. DUE-B therefore shows strong similarity to the yeast Cdc45-loading protein Sld3, a key target of the S phase promoting cyclin- dependent kinases. We propose to test a model in which metazoan DUE-B is the functional homolog of Sld3. Adding significance to the role of DUE B in replication, we have found a strong correlation between DUE-B overexpression and ovarian cancer. Remarkably, DUE-B also shows a second personality;the structure of the N-terminal 75% of the protein has been strongly conserved during evolution, and displays aminoacyl-tRNA proofreading activities found in yeast, bacteria and archae. We will use HeLa cells and Xenopus egg extracts to address several mechanistic aspects of the model in which DUE-B regulates the binding of the helicase activator Cdc45 to form the replication preinitiation complex, and in which the DUE-B-dependent loading of Cdc45 is a target of the intra-S phase DNA damage checkpoint. Our major analytical techniques will be immunoblotting, chromatin immunoprecipitation, quantitative PCR, FRET, and immunofluorescence. In Aim 1 we will analyze the binding of DUE-B and preinitiation complex proteins to replication origins, and the effect of the intra-S phase DNA damage checkpoint on DUE-B function. Aim 2 will characterize the effects of targeted structural mutations in DUE-B on its binding to protein ligands and its activation of replication origins.